Abstract
Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens, Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-D-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit anti-biofilm activity, and further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted pre-formed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h. In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were non-cytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 hours. Intratracheal administration of Sph3h was well tolerated, and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.
Significance The production of biofilms is an important strategy used by both bacteria and fungi to colonize surfaces and to enhance resistance to killing by immune cells and antimicrobial agents. We demonstrate that glycoside hydrolases derived from the opportunistic fungus Aspergillus fumigatus and Gram-negative bacterium Pseudomonas aeruginosa can be exploited to disrupt pre-formed fungal biofilms and reduce virulence. Additionally, these glycoside hydrolases can be utilized to potentiate antifungal drugs by increasing their hyphal penetration, to protect human cells from fungal-induced injury and to attenuate virulence of A. fumigatus in a mouse model of invasive aspergillosis. The findings of this study identify recombinant microbial glycoside hydrolases as promising therapeutics with the potential for anti-biofilm activity against pathogens across different taxonomic kingdoms.
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The mould Aspergillus fumigatus and the Gram-negative bacterium Pseudomonas aeruginosa are opportunistic pathogens that cause pulmonary infection in immunocompromised patients and individuals who suffer from chronic lung diseases such as cystic fibrosis and bronchiectasis. A. fumigatus is the second most common nosocomial fungal infection (1) and ~10% of all nosocomial bacterial infections are caused by P. aeruginosa (2). Mortality associated with P. aeruginosa infections is high (3), and has increased with the emergence of multi- and even pan-resistance to antibiotics (3, 4). Similarly, invasive aspergillosis is associated with mortality rates of up to 50% (5), and increasing rates of antifungal resistance have been reported worldwide (6). These factors underscore the urgent need for new effective therapies for these infections.
Although A. fumigatus and P. aeruginosa are members of different taxonomic kingdoms, both produce biofilms that constitute a protective lifestyle for the organism. Biofilms are complex communities of microorganisms that grow embedded in an extracellular matrix composed of DNA, protein, and exopolysaccharide (7). Biofilm formation provides a significant advantage to these organisms as the matrix mediates adherence to host cells (8, 9) and aids in the resistance to both antimicrobial agents (10, 11) and host immune defences (12, 13). A. fumigatus biofilm formation is dependent on the cationic polysaccharide galactosaminogalactan (GAG), a heteroglycan composed of α1,4-linked galactose and N-acetyl-D-galactosamine (GalNAc) that is partially deacetylated (14, 15). In comparison, P. aeruginosa has the genetic capacity to produce three biofilm exopolysaccharides; alginate, Psl and Pel (16). GAG shares several similarities with Pel, which has been identified as a cationic heteroglycan composed of 1,4-linked GalNAc and N-acetyl-D-glucosamine (GlcNAc) (17). Like GAG, the cationic nature of Pel results from partial deacetylation of the polymer (17). Most clinical and environmental isolates of P. aeruginosa utilize Pel and Psl during biofilm formation (18). Alginate is dispensable for biofilm formation and is only observed in chronic pulmonary infection when strains switch to a mucoid phenotype (18, 19).
Strains of Aspergillus and P. aeruginosa with impaired GAG, or Pel and Psl biosynthesis exhibit attenuated virulence (20, 21), suggesting that targeting these exopolysaccharides may be a useful therapeutic strategy. We previously demonstrated that recombinant glycoside hydrolases PelAh and PslGh, encoded in the pel and psl operons of P. aeruginosa, respectively, target and selectively hydrolyze the Pel and Psl exopolysaccharide components of the Pseudomonas biofilm matrix (22). Treatment with these enzymes rapidly disrupts established biofilms, increasing the susceptibility of P. aeruginosa to human neutrophil killing and potentiation of the antibiotic colistin (22).
Our recent work on Aspergillus has identified a cluster of five genes, which encode the proteins necessary for GAG biosynthesis (15). As with P. aeruginosa, we found that the product of one of these genes contains a glycoside hydrolase domain, Sph3h, that is capable of hydrolyzing purified and cell wall-associated GAG (23). In the present study we assessed the therapeutic potential of Sph3h in disrupting fungal biofilms. We establish that the exogenous addition of Sph3h is capable of rapidly disrupting existing biofilms of this organism at nanomolar concentrations. Additionally, we demonstrate cross-kingdom activity, as the P. aeruginosa glycoside hydrolase, PelAh, was able to disrupt A. fumigatus biofilms. While Sph3h was able to bind Pel, it was unable to disrupt pre-formed P. aeruginosa Pel-mediated biofilms. Treatment with Sph3h or PelAh increased the susceptibility of wild-type and azole-resistant A. fumigatus strains to lipophilic antifungal drugs. Kinetic studies with labelled posaconazole indicate that the increased susceptibility to antifungals is due to increased penetration of fungal cells by these agents. Both Sph3h and PelAh were non-toxic to mammalian cells and protected epithelial cells from A. fumigatus-induced damage for up to 24 hours. Intratracheal delivery of Sph3h was well tolerated by mice and significantly reduced the fungal burden of immunocompromised mice infected with A. fumigatus. Our results suggest that glycoside hydrolases have the potential to be effective anti-biofilm therapeutics that can mediate activity against evolutionarily diverse microorganisms.
Results
Sph3h disrupts preformed A. fumigatus biofilms
Our previous work demonstrated that Sph3h from A. fumigatus and Aspergillus clavatus can hydrolyze both purified and cell wall-bound GAG on young hyphae (23). We therefore sought to determine if the degradation of GAG by Sph3h could disrupt established A. fumigatus biofilms. Treatment with Sph3h for one hour disrupted established A. fumigatus biofilms with an effective concentration for 50% activity (EC50) of 0.45 ± 1.31 nM (Fig. 1a). Biofilm disruption was associated with a marked reduction in hyphae-associated GAG as detected by lectin staining (Fig. 1b and c) and scanning electron microscopy (Fig. 1d). A catalytic variant Sph3h D166A, which does not mediate GAG hydrolysis (23), displayed a greater than 500-fold reduction in anti-biofilm activity (Fig. 1a) and failed to mediate degradation of biofilm-associated GAG (Fig. 1b and c). Collectively, these data suggest that biofilm disruption is mediated through the enzymatic hydrolysis of GAG.
To validate that fungal biofilm disruption by Sph3h is not restricted to the A. fumigatus laboratory strain Af293, the activity of Sph3h was evaluated against four clinical A. fumigatus isolates. Sph3h disrupted biofilms of all isolates tested at EC50 values < 0.15 nM (Fig. S1). These results confirm the role of GAG in biofilm formation and indicate that Sph3h exhibits anti-biofilm activity across a wide range of A. fumigatus strains.
The bacterial hydrolase PelAh hydrolyses GAG and disrupts fungal biofilms
Given that GAG and Pel are both cationic exopolysaccharides containing 1,4-linked GalNAc (14, 17), we hypothesized that PelAh might exhibit activity against GAG. Consistent with this hypothesis, an in vitro reducing sugar assay demonstrated that PelAh was capable of hydrolyzing purified GAG (Fig. S2). Furthermore, using the crystal violet biofilm assay, we found that PelAh disrupted A. fumigatus fungal biofilms with an EC50 value of 2.80 ± 1.14 nM (Fig. 2a). The treatment of A. fumigatus hyphae with PelAh also resulted in a reduction in the amount of cell wall-associated GAG (Fig. 2b-d) as was observed with Sph3h treatment. The PelAh catalytic variant, PelAh E218A, which is markedly impaired in Pel hydrolysis and is inactive against P. aeruginosa biofilms (22) did not significantly hydrolyze GAG at concentrations as high as 12 μM (Fig. S2). Consistent with this observation, PelAh E218A was also several hundred-fold less active against A. fumigatus biofilms and did not degrade hyphae-associated GAG (Fig. 2b and c). These results suggest that PelAh disrupts A. fumigatus biofilms through the hydrolysis of biofilm associated GAG.
Sph3h binds Pel but does not disrupt established P. aeruginosa biofilms
Given that PelAh can hydrolyze GAG and disrupt GAG-mediated biofilms, we hypothesized that Sph3h may exhibit activity against Pel and Pel-mediated biofilms. The inability to purify sufficient quantities of Pel precluded us from utilizing it as a substrate. Therefore, to examine whether Sph3h was capable of hydrolyzing Pel, the enzyme was exogenously applied to biofilms produced by the Pel overproducing P. aeruginosa strain PAO1 ΔwspF Δpsl PBADpel. Treatment of these established biofilms with Sph3h did not affect levels of Pel within the biofilms as visualized by lectin staining (Fig. 3a and b), nor did it reduce biofilm biomass, even at concentrations exceeding 10 μM (Fig. 3c).
Since Sph3h did not hydrolyze Pel within P. aeruginosa biofilms, we tested whether the enzyme was capable of recognizing and binding this polysaccharide. Using an ELISA-based binding assay we observed dose-dependent binding of Sph3h to culture supernatants from the Pel over-producing P. aeruginosa strain, but not from supernatants of the Pel-deficient strain PAO1 ΔwspF Δpel Δpsl (Fig. 3d). These data suggest that the inability of Sph3h to disrupt Pel-mediated biofilms is likely a consequence of an inability to hydrolyze Pel rather than being unable to bind the polysaccharide. Dose-dependent binding of the inactive Sph3h D166A variant to GAG-containing culture supernatants was also observed (Fig. S3), suggesting that binding of hydrolases to exopolysaccharides is insufficient to disrupt established biofilms in the absence of enzymatic cleavage of the polymer.
Sph3h and PelAh potentiate antifungals by enhancing their intracellular penetration
The Pel polysaccharide enhances resistance to several antibiotics including aminoglycosides and colistin (22, 24,25). Since biofilm formation by A. fumigatus is associated with increased resistance to a number of antifungal agents (26–28), we hypothesized that GAG may have an analogous function to Pel in enhancing resistance to antifungal agents. To test this hypothesis, we investigated whether Sph3h or PelAh could potentiate the activity of commonly used antifungal drugs. Treatment of established fungal biofilms with either enzyme resulted in a significant reduction in the MIC50 of the azole posaconazole, the polyene amphotericin B, and the echinocandin caspofungin (Fig. 4a). Sph3h or PelAh treatment produced a similar increase in sensitivity to posaconazole for both azole-sensitive and azole-resistant strains of A. fumigatus (Fig. S4). Susceptibility to voriconazole, a smaller and more polar azole, was unaffected by treatment with either glycoside hydrolase (Fig. 4a). Since both posaconazole and voriconazole have the same intracellular target, these findings suggest that cationic GAG mediates antifungal resistance by hindering cellular uptake of large, nonpolar molecules such as posaconazole. To investigate this hypothesis, the effect of Sph3h on intracellular penetration of posaconazole was examined using posaconazole conjugated to the fluorophore BODIPY (BDP-PCZ). Previous work has established that BDP-PCZ displays similar cellular and subcellular pharmacokinetics to unmodified posaconazole (29). Fluorometric studies revealed that Sph3h-treatment resulted in higher accumulation of BDP-PCZ within A. fumigatus hyphae (Fig. 4b). This finding indicates that GAG protects A. fumigatus from the action of lipophilic antifungals by limiting their penetration into hyphae.
Recombinant Sph3h and PelAh protect epithelial cells from damage by A. fumigatus
A. fumigatus GAG-mediated adherence is required for A. fumigatus to damage A549 pulmonary epithelial cells in vitro (20). We therefore tested whether treatment with either Sph3h or PelAh could protect epithelial cells from fungal-induced injury using an established chromium (51Cr) release damage assay (30). We first established that the enzymes were not cytotoxic and that the addition of Sph3h or PelAh to uninfected A549 cell monolayers did not cause detectable cellular damage (Fig. S5a), a finding verified with the IMR-90 human lung fibroblast cell line (Fig. S5b). These data are consistent with the lack of cytotoxicity previously reported for PelAh (22). Next, we assessed whether Sph3h or PelAh were able to protect A549 cell monolayers from damage by A. fumigatus. Sph3h reduced epithelial cell injury by > 80% for 24 hours (Fig. 5a). Treatment with PelAh also protected epithelial cells from A. fumigatus-induced damage (Fig. 5a). The protective effect of PelAh was shorter than that observed with Sph3h, and was lost before 24 hours of treatment. The addition of protease inhibitors extended PelAh- mediated epithelial cell protection to 24 hours (Fig. S5c), suggesting that the decrease in PelAh mediated protection was likely due to proteolytic degradation of the recombinant protein. Epithelial cell protection was not observed with the catalytic variants, PelAh E218A or Sph3h D166A, suggesting that the hydrolytic activity of the enzymes is required for protection (Fig 5a).
Intratracheal Sph3h is well tolerated, and attenuates fungal virulence in an immunocompromised mouse model of pulmonary aspergillosis
Given the ability of Sph3h to protect epithelial cells for over 24 hours, this hydrolase was selected for evaluation in vivo. BALB/c mice treated intratracheally with doses up to 500 μg of Sph3h exhibited no signs of stress, weight loss or change in body temperature post-treatment (Fig. S6a and b). Additionally, no significant increase in pulmonary injury or inflammation between treated and untreated mice were observed as measured by bronchoalveolar lavage lactate dehydrogenase activity and total pulmonary leukocyte populations (Fig. 5b and Fig. S6c). Collectively these results suggest that a single intratracheal dose of Sph3h is well tolerated by mice.
To determine the ability of Sph3h to attenuate virulence of A. fumigatus, neutropenic BALB/c mice were infected intratracheally with A. fumigatus conidia with or without the co-administration of 500 μg of Sph3h. Four days after infection, mice infected with A. fumigatus and treated with Sph3h had a significantly lower pulmonary fungal burden to untreated, infected mice as measured by both fungal DNA (Fig. 5c) and pulmonary galactomannan content (Fig. S7). The fungal burden of the Sph3h-treated mice was similar to that observed with mice infected with the GAG-deficient hypovirulent strain Δuge3 (20). Consistent with the fungal burden data, histopathologic examination of lung sections revealed the presence of fungal lesions in untreated, infected mice, but no detectable lesions in the lungs of infected mice treated with Sph3h, or those infected with conidia of the Δuge3 mutant (Fig. 5d). These findings suggest that Sph3h-mediated degradation of GAG can limit the growth of A. fumigatus in vivo, to the same degree as is observed with GAG-deficient organisms.
Discussion
In this study, we demonstrate that the fungal glycoside hydrolase Sph3h is able to degrade pre-formed A. fumigatus biofilms. This study is the first example of the use of a glycoside hydrolase to disrupt a fungal biofilm. Further, we establish that the glycoside hydrolase PelAh displays activity against biofilms formed by organisms across different microbial kingdoms. Both glycoside hydrolases potentiated the penetration and activity of antifungal agents in vitro, exhibited no toxicity against mammalian cells and protected epithelial cells from A. fumigatus-induced damage. Pulmonary administration of Sph3h was well tolerated and limited fungal growth in an immunocompromised mouse model, suggesting that these enzymes are promising therapeutic agents for the treatment of fungal disease.
The mechanism by which the biofilm matrix enhances A. fumigatus resistance to antifungals is poorly understood. The effect of hydrolase treatment on the antifungal sensitivity of A. fumigatus provides some insight into this question and establishes a role for GAG in biofilm-associated antifungal resistance. Multiple observations suggest that GAG enhances antifungal resistance by acting as a barrier to antifungal penetration of hyphae. First, glycoside hydrolase degradation of GAG enhanced the activity of multiple antifungals with different mechanisms of action. Second, the activity of posaconazole, but not voriconazole, was enhanced even though both azoles target the same enzyme, CYP51A. These hydrolases also display similar activity against azole-resistant and azole-sensitive strains. The cationic nature of GAG may explain the differential effects on voriconazole as compared with other antifungals. The GAG barrier would be predicted to be most effective against large, lipophilic or cationic antimicrobial agents, and thus therapeutic hydrolases may be most effective as adjuvants for lipophilic antifungals. Previous studies have reported that the enzymatic degradation of neutral α-glucans of A. fumigatus did not enhance susceptibility to antifungals (31), further supporting our hypothesis that exopolysaccharide charge plays a role in mediating antibiotic resistance. Similarly, hydrolysis of cationic Pel exopolysaccharide by PelAh enhances the activity of the polycationic antibacterial colistin (22). Interestingly, degradation of biofilm-associated extracellular DNA (eDNA) has previously been reported to enhance A. fumigatus susceptibility to caspofungin and amphotericin B, though the effects on posaconazole and voriconazole susceptibility were not reported in the study (26). Recent work has suggested that Pel anchors eDNA within P. aeruginosa biofilms through charge-charge interactions (17). Given the similarities between Pel and GAG, it is possible that GAG-mediated binding of eDNA may also contribute to enhancing antifungal resistance.
The results of these studies add to an emerging body of evidence that fungal biofilms share structural (32–35) and functional (26, 36,37) similarity with those formed by pathogenic bacteria. The finding that glycoside hydrolases can display activity against the exopolysaccharides and biofilms of both fungi and bacteria provides the first evidence that these similarities could potentially be exploited for the development of therapeutics active against both organisms. Additionally, the similarity between the exopolysaccharides of P. aeruginosa and A. fumigatus, coupled with the interspecies activity of their glycoside hydrolases suggest the intriguing possibility that exopolysaccharide interactions may occur between organisms during multispecies biofilm formation. Co-colonization with P. aeruginosa and A. fumigatus is not uncommon in patients with chronic pulmonary disease such as cystic fibrosis (38). Although studies of the formation of mixed fungal-bacterial biofilms during pulmonary infection are limited, a recent study of patients with chronic lung disease reported that antibacterial therapy for P. aeruginosa was associated with a reduction in fungal colonization, suggesting the possibility of microbial cooperation (39). Further studies examining the role of cross-species exopolysaccharide and exopolysaccharide-modifying enzyme interactions are required to establish a role for cooperative biofilm interactions in pulmonary disease.
While PelAh exhibited cross-species activity and disrupted pre-formed fungal biofilms, Sph3h bound Pel, but was unable to disrupt established Pel-mediated biofilms. This difference in activity may reflect differences in the composition or conformation of each polysaccharide since GAG is a heteropolymer of GalNAc and galactose while Pel is comprised of GalNAc and GlcNAc. It is likely that these differences influence the ability of Sph3h and PelAh to hydrolyze the polymer. The inability of Sph3h to degrade preformed P. aeruginosa biofilms may suggest that mature Pel adopts a configuration or undergoes post-synthetic modification that renders it incompatible with the catalytic active site of Sph3h and resistant to cleavage. Detailed studies of these enzymes to determine the mechanisms underlying their differential activity against Pel will require purified polysaccharide, which is currently not available.
Both Sph3h and PelAh were found to be non-cytotoxic, and a single dose of intratracheal Sph3h was well tolerated by BALB/c mice. Co-administration of Sph3h with wild-type conidia to neutropenic mice greatly reduced fungal outgrowth within the lungs of these mice. Together these results provide proof-of-concept that the glycoside hydrolases can be used to improve the outcome of fungal infection, with minimal side effects and toxicity. These findings will pave the way for future work to evaluate the utility of these agents as antifungal therapeutics including detailed pharmacokinetic and toxicity studies, as well as the evaluation of these enzymes for the treatment of established fungal infections alone and in combination therapy with lipophilic antifungal agents such as posaconazole or amphotericin B.
Methods
Strains and culture conditions
Strains used in this study are detailed in Table S1 and detailed culture conditions are described in the Supplementary Information (SI). Recombinant hydrolase expression and purification. Hydrolases were expressed and purified as described previously (22, 23).
Treatment of A. fumigatus with glycoside hydrolases
To visualize the effects of hydrolases on cell wall-associated GAG, hyphae were treated with recombinant hydrolases and stained with fluorescein-conjugated soybean agglutinin as previously described (23), with minor modifications. Hyphae were counterstained with a 1:1000 dilution of DRAQ5 (eBioscience) in phosphate buffered saline (PBS) for 5 min prior to paraformaldehyde (PFA) fixation. Complete image acquisition and processing methods can be found in the SI. To study the effects of hydrolases on biofilms, 104 conidia were grown in Brian media in polystyrene, 96-well plates for 19 h at 37 °C and 5% CO2 and then treated with the indicated concentration of glycoside hydrolase in PBS for 1 h at room temperature. Biofilms were then gently washed, stained with 0.1% (w/v) crystal violet and de-stained with 100% ethanol for 10 min. The optical density of the de-stain fluid was measured at 600 nm (OD600).
Scanning electron microscopy
Conidia were grown for 9 h in Dulbecco's Modified Eagle's Medium (DMEM) at 37 °C, 5% CO2 on glass coverslips, washed once with Ham's F- 12K (Kaighn's) Medium, and incubated with 500 in F-12K Medium with or without 0.5 μM hydrolase for 3 h at 37°C, 5% CO2. Coverslips were processed for scanning electron microscopy as previously described (20), and detailed in the SI.
Treatment of P. aeruginosa with glycoside hydrolases
For biofilm disruption, static P. aeruginosa cultures were grown for 22 h at 30 °C, at which point the planktonic cells were aspirated and LB + 0.5% arabinose + 0.5 μM glycoside hydrolase was added for an additional 3 h. For the detection of Pel, samples were incubated with 30 μg/ml of fluorescein-conjugated Wisteria fluoribunda lectin for 2 h at 4 °C, fixed with 8% (w/v) PFA for 20 min at 4° C and imaged as detailed in the SI. The ability of hydrolases to disrupt established biofilms were studied as previously described (22).
Culture supernatant production
P. aeruginosa cultures were grown at 30 °C for 24 h shaking at 200 rpm. Cultures were then centrifuged at 311 x g for 10 mins, and supernatants were filtered using 0.44 μm syringe filters. Culture supernatants were stored at −20 °C until use.
Hydrolase binding quantification
Undiluted culture supernatants were incubated on Immunolon® 2HB high-binding 96 well microtiter plates overnight at 4 °C. Wells were washed 3X with Wash Buffer (PBS + 0.05% (v/v) Tween-20) and blocked for 30 min at 4 °C in Blocking Buffer (1% (w/v) Bovine Serum Albumin in Wash Buffer). Wells were washed 1X with Wash Buffer and incubated with the indicated concentrations of Sph3h diluted in Blocking Buffer for 3 h at 4 °C. Wells were washed 3X with Wash Buffer and incubated with polyclonal rabbit anti-Sph3h (custom-produced by Cedarlane Laboratories) diluted 1/100 in Blocking Buffer for 1.5 h at 4 °C. Wells were then washed 3X with Wash Buffer and incubated with donkey-anti-rabbit secondary antibody conjugated to horseradish peroxidase diluted 1/2000 in Blocking Buffer for 1 h at 4 °C. Wells were then washed 4X with Wash Buffer and incubated with TMB substrate (ThermoFisher®) for 15 mins at room temperature. The reaction was stopped with the addition of 2 N H2SO4 and absorbance read at 450 nm with a 570 nm correction.
Effects of glycoside hydrolases on antifungal susceptibility of A. fumigatus.
Fungal biofilms were prepared in tissue culture treated 24 well plates in RPMI 1640 medium (Life Technology) buffered with MOPS (3-(N-Morpholino) Propane-Sulfonic Acid) (Fisher) (RPMI-MOPS) for 9 h at 37 °C, 5% CO2. Serial dilutions of antifungal compounds with or without 0.5 μM of Sph3h or PelAh were added to wells and the plates incubated at 37 °C and 5% CO2 for 15 h. Fungal viability was measured using the sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene-sulfonic acid hydrate (XTT) metabolic assay as described previously (40). The concentration of antifungal resulting in a 50% decrease in viability (MIC50) was used as a measure of antifungal effect.
Fluorometric quantification of hyphal uptake of BDP-PCZ
2.5x104 conidia of red fluorescent protein (RFP)-expressing A. fumigatus were grown in a 96-well black, clear-bottom plate for 8 h at 37 °C, 5% CO2. Hyphae were treated with 1 μM of Sph3h in PBS for 90 min at 37 °C, 5% CO2 then exposed to 2 μg/mL BDP-PCZ for 10 mins. The plate was then read using an Infinite M1000 fluorescent plate reader with excitation wavelengths of 532 and 488 nm for RFP and BDP-PCZ, respectively. Background fluorescence was subtracted from both RFP and BDP-PCZ signals and the BDP-PCZ signal was then normalized to total RFP fluorescence for each well.
Effects of glycoside hydrolases on A. fumigatus-induced epithelial cell damage
A549 pulmonary epithelial cell damage by A. fumigatus was tested using the 51Cr release assay as previously described (20, 30). Recombinant hydrolases were added to the A549 cultures at the time of infection at a final concentration of 0.5 μM.
Characterization of pulmonary damage by Sph3h
All procedures involving mice were approved by the Animal Care Committees of the McGill University Health Centre. Female BALB/c mice 5-6 weeks of age were anaesthetized with isoflurane and administered a single endotracheal injection of 500 μg Sph3h in 50 μL PBS and monitored daily for 7 days. Mice were then euthanized by CO2 overdose and their airways lavaged with 1 mL PBS that was administered and collected through a needle inserted in the trachea. A total of 2 lavages were performed and pooled. The presence of LDH in the BAL fluid was used as an indicator of pulmonary damage; LDH activity was measured in the fluid using a commercial assay (Promega), as per manufacturer’s instructions.
Effects of Sph3h in a severely immunocompromised mouse model of invasive pulmonary aspergillosis
Mice were immunosuppressed with cortisone acetate and cyclophosphamide as previously described (20, 41). Mice were infected with an endotracheal injection of 5x103 A. fumigatus conidia, resuspended in either PBS alone, or in combination with 500 μg of Sph3h. Mice were monitored daily and moribund animal were euthanized. At 4 days post infection mice were euthanized and their lungs were harvested. For fungal burden analysis, lungs were homogenized in 5 mL PBS containing protease inhibitor cocktail (Roche), and aliquots were stored at −80°C until use. Pulmonary fungal burden was determined as previously described (15), and detailed in the SI. For histological examination, lungs were inflated with 10% buffered formalin (Fisher Scientific) and immersed in formalin overnight to fix. Lungs were then embedded in paraffin and 4 μm thick sections were stained with periodic acid Schiff (PAS) stain.
Acknowledgements
Research described in this paper is supported by operating grants from the Canadian Institutes of Health Research (CIHR) (#81361 to P.L.H. and D.C.S; #123306 to D.C.S.; and #43998 to P.L.H) and Cystic Fibrosis Canada (CFC) (to D.C.S. and P.L.H). B.D.S has been supported by graduate scholarships from CFC and CIHR. N.C.B has been supported in part by graduate scholarships from the Natural Sciences and Engineering Research Council of Canada, Mary H. Beatty, and Dr. James A. and Connie P. Dickson Scholarships from the University of Toronto, CFC, and The Hospital for Sick Children. P.B. has been supported in part by a CFC postdoctoral fellowship and a Banting Fellowship from CIHR. P.L.H. is the recipient of a Canada Research Chair. D.C.S is supported by a Chercheur-Boursier Award from the Fonds de Recherche Quebec Santé.